Therapeutic peptides are used to treat human diseases ranging from HIV to diabetes and have some of the best features of small molecule and recombinant protein drugs. Therapeutic peptides account for $13 billion of annual pharmaceutical sales and are part of a growing sector of the biopharmaceutical market. Unfortunately, therapeutic peptides suffer from poor stability and short half-lives in the human body, which limits their value. The requirement for high dosing and frequent injections can be inconvenient, expensive, and dangerous for patients. While there have been methods developed to address these issues, they either: (i) hinge on the in vitro or recombinant attachment of a large polymer chain, which dramatically impacts peptide activity or (ii) require in vitro processing steps which increase manufacturing costs and complicate purification. It is now well- established that the stability and half-life of peptide drugs can be greatly improved by conjugation to humanlike oligosaccharides. Several therapeutic peptides (e.g., Exenatide, Glucagon-like peptide 1) have benefitted significantly from glycosylation with small human-like glycans by increasing protease resistance, prolonging activity, and improving biodistribution. However, this requires multiple complicated in vitro reactions and purifications which have kept this promising concept from reaching the industrial scale. At Glycobia we have developed novel strains of Escherichia coli for the expression of recombinant peptides conjugated to humanlike oligosaccharides. In Phase I of this project, we applied our glycoengineered bacteria as a platform for the biosynthesis of therapeutic glycopeptides by: (1) expressing a panel of therapeutic peptide glycoconjugates and screening for glycosylation efficiency and (2) screening glycoconjugate drug candidates for physical properties and in vitro activity. We show proof-of-concept of several recombinant peptides with improved stability and/or activity when modified with glycosylation. Now having identified lead candidates for Phase II of this project, the objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E. coli into preclinical testing by: (1) expressing, scaling up, purifying, and characterizing a glycosylated human peptide drug from in E. coli and (2) testing pharmacology, calcemic response and pharmacokinetics of a glycosylated human peptide drug in animal models. We will attach two different humanlike glycans to the drug and compare performance to an aglycosylated version of the drug. The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this glycoengineering technology. Our bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of producing improved therapeutic peptides for patients.